Agglutination immunoassay and kit for determination of a multivalent immune species using a buffered salt wash solution

Abstract

A test kit is used in an agglutination immunoassay to determine a multivalent immune species, such as Streptococcus A antigen, in a biological sample. The method includes contacting an aqueous solution of the species with an agglutination indicator reagent having receptor molecules reactive with the species to form an agglutinate of the reaction product of species and receptor. These receptor molecules are bound to polymeric particles which contain tracer molecules. The resulting agglutinate is captured on a microporous membrane which has an average pore size which is at least five times greater than the average diameter of the polymeric particles. Unagglutinated residual materials are washed through the membrane using a wash solution. This solution has a pH of from about 5 to about 10 and an ionic strength of at least about 0.25. Tracer is then determined either in the agglutinate or in the residual materials. The test kit includes the agglutination indicator reagent, the wash solution, and optionally an extraction composition.

We claim: 
     
       1. An agglutination method for the determination of a multivalent immune species comprising: (a) contacting an aqueous liquid containing said species in free form with a reagent comprising water-insoluble particles having tracer molecules associated therewith and receptor molecules reactive with said species bound to a surface thereof, so as to form an agglutinate of a reaction product of said species and said receptor molecules,   (b) simultaneously or subsequent to said contacting step (a), contacting said agglutinate with a microporous water-insoluble membrane having an average pore size which is at least 5 times greater than the average diameter of said water-insoluble particles,   (c) washing unagglutinated residual materials through said membrane while leaving said agglutinate thereon, said washing accomplished with a wash solution having a pH of from about 5 to about 10 and an ionic strength of at least about 0.25, and   (d) determining the amount of tracer molecules either in said agglutinate remaining on said membrane or said residual materials.   
     
     
       2. The method of claim 1 wherein said wash solution has an ionic strength of from about 0.5 to about 3. 
     
     
       3. The method of claim 1 wherein said wash solution has a pH of from about 6 to about 9. 
     
     
       4. The method of claim 1 wherein said species is extracted from a biological sample with an extraction composition prior to said contacting step (a). 
     
     
       5. The method of claim 1 wherein said contacting step (a) is carried out simultaneously with contact with said microporous membrane. 
     
     
       6. The method of claim 1 for the determination of an antigen wherein said receptor molecules are antibodies for said antigen which are covalently bound to said water-insoluble particles. 
     
     
       7. The method of claim 1 for the determination of antibodies wherein said receptor molecules are antigen molecules reactive with said antibodies. 
     
     
       8. The method of claim 1 wherein said water-insoluble particles contain molecules of a colorimetric dye as tracer molecules. 
     
     
       9. The method of claim 1 wherein said tracer molecules are rare earth chelate molecules. 
     
     
       10. The method of claim 1 wherein said tracer molecules are determined in said agglutinate on said membrane. 
     
     
       11. The method of claim 1 wherein said water-insoluble particles have an average diameter of less than about 1 micrometer. 
     
     
       12. The method of claim 11 carried out using a disposable test device containing said microporous membrane. 
     
     
       13. An agglutination method for the determination of Streptococcus A in a biological sample comprising: (a) extracting Streptococcus A antigen from said sample with an extraction composition,   (b) contacting said extracted antigen with a reagent comprising water-insoluble, polymeric latex particles having tracer molecules distributed therein and antibodies reactive with said extracted antigen covalently bound to a surface thereof, so as to form an agglutinate of a reaction product of said antigen and said antibodies, said contacting being carried out in an aqueous mixture in the presence of a microporous water-insoluble membrane having an average pore size which is at least five times greater than the average diameter of said water-insoluble particles,   (c) washing unagglutinated residual materials through said membrane while leaving said agglutinate thereon, said washing accomplished with a wash solution having a pH of from about 6 to about 9 and an ionic strength from about 0.25 to about 3, and   (d) determining an amount of said tracer molecules in said agglutinate remaining on said membrane.   
     
     
       14. The method of claim 13 wherein said extraction is carried out with an extraction composition comprising a nitrite salt and citric acid. 
     
     
       15. The method of claim 13 wherein said polymeric latex particles are core-shell latex particles containing substantially all of said tracer molecules in a particle core and having an average diameter of from about 0.1 to about 0.7 micrometer. 
     
     
       16. The method of claim 13 wherein said tracer molecules are colorimetric dye molecules. 
     
     
       17. A method for detecting Streptococcus A comprising: (i) providing an applicator including an applicator stick and a fibrous swab and collecting a biological sample with said swab,   (ii) providing an extraction composition comprising sodium nitrite and citric acid for effecting release of Streptococcus A antigen from said swab, dipping said swab in said extraction composition and incubating said swab within said extraction composition for a period of time sufficient to release antigen from said swab,   (iii) neutralizing the solution of said extracted antigen,   (iv) contacting said neutralized solution of said extracted antigen with an agglutination indicator reagent comprising water-insoluble, polymeric core-shell latex particles having an average diameter of from about 0.1 to about 0.7 micrometer, and having molecules of a colorimetric dye distributed substantially in particle cores and comprising antibodies reactive with said antigen bound to the surface thereof, so as to form an agglutinate of a reaction product of said antigen and said antibodies, said contacting being carried out in the presence of a microporous water-insoluble membrane mounted in a disposable test device, said membrane having an average pore size which is at least 5 times greater than an average diameter of said water-insoluble particles   (v) washing unagglutinated residual materials through said membrane while leaving said agglutinate thereon, said washing accomplished with an aqueous wash solution having a pH of from about 6 to about 9 and an ionic strength of from about 0.5 to about 3, and   (vi) determining the amount of colorimetric dye molecules in said agglutinate remaining on said membrane.   
     
     
       18. The method of claim 17 wherein said aqueous wash solution comprises sodium chloride. 
     
     
       19. A test kit for the determination of a multivalent immune species comprising: an agglutination indicator reagent comprising water-insoluble particles having tracer molecules associated therewith and receptor molecules reactive with said species bound to the surface thereof, and   a wash solution having a pH of from about 5 to about 10 and an ionic strength of at least about 0.25.   
     
     
       20. The test kit of claim 19 for the determination of Streptococcus A antigen further comprising: an applicator means for collecting a biological sample suspected of containing Streptococcus A, said applicator means including an applicator stick and a swab at one end thereof that collects said sample, and   an extraction composition for extracting Streptococcus A antigen from Streptococcus A present in said sample.   
     
     
       21. The test kit of claim 19 further comprising a disposable test device comprising a microporous water-soluble membrane mounted in a test well of said test device, said membrane having an average pore size which is at least five times greater than the average diameter of said water-insoluble particles.